大腸桿菌ClpYQ蛋白酶之ClpY I domain區域双環構造與孔洞區所扮演角色

Abstract

摘要 ATP依賴型蛋白酶對於控制關鍵調控蛋白表現量及降解異常蛋白質以維持微生物體內正常生理活動扮演著重要的角色。ClpYQ蛋白酶為ATP依賴型蛋白酶其中的一種由ClpY與ClpQ二種次單元體所組成。ClpY具有ATPase與chaperone的功能而ClpQ為一胜肽酶。ClpY可以自身形成的六元環而與ClpQ十二元體上下相接形成啞鈴狀的聚合分子。ClpY主要負責辨識、解構與運送基質SulA至ClpQ十二元體的活性中心進行分解。ClpY單元體由N、 I 與C三個domain組成各有其獨特的活性而I domain區域突出於N與C domain之外，其主要的功能尚未有清楚的界定。本研究發現ClpYQ蛋白酶對基質MBP-SulA的辨識結合主要是由loop2結構來進行。而I domain loop1(aa.137-150) 結構則是可能在ClpY在與MBP-SulA辨識結合後，ClpYQ蛋白酶進行降解作用時，阻擋多餘的MBP-SulA與ClpY六元環結合的功能。在pore I/loop2雙缺失突變蛋白ΔP1L2(aa 90-93；aa 175-209)在in vitro pull down實驗中有與MBP-SulA最低的相對活性，顯示基質的辨識結合由pore I與loop2共同負責。另外ClpY可以在沒有ATP存在下與SulA進行辨識結合，而且當ClpY形成六元環後只能與少量的MBP-SulA結合。綜合以上結果可推論出ClpY與SulA進行辨識結合的步驟：即ClpY在沒有ATP存在下與SulA進行辨識結合後，SulA-ClpY在ATP存在下與其他未結合的ClpY形成SulA-ClpY6或SulA-ClpY12的聚合，再與ClpQ十二元體形成SulA-ClpY6Q12聚合體，再進行解構、傳送及降解的步驟。而ClpYY408A突變蛋白無法形成穩定的六元環及ClpYQ聚合體，但仍保有部分的ATPase活性及部分的降解活性，顯示了ClpYQ聚合的穩定度對酵素的活性是重要的。

Abstract ATP dependent proteases play important roles in controlling the levels of key regulatory protein and in the elimination of abnormal proteins to maintain normal physiological functions of microorgamisms. ClpYQ protease, one of ATP dependent proteases includes two subunits ClpY and ClpQ. ClpY acts as an ATPase and chaperone and ClpQ is a peptidase. ClpY is capble of forming a hexamer ring docked with ClpQ dodecamer to constitute a dumbbel-shaped complex. ClpY is responsible to recognize, unfold and traslocate substractes into the proteolytic site of ClpQ for degradation. Besides, ClpY is divided into three domains N, I and C domain. Each domain has it’s own distinct activity. I domain, a unique protruding domain of ClpY, is unclear for its function. In this study, our results demonstrated that ClpY I domain loop2 is responsible for the initial gripping of SulA and loop1 acts as a lid that is likely to prevent an excess of substracts binding for ClpY when ATP is present. ΔP1L2(aa 90-93；aa 175-209) showed the lowest binding activity with MBP-SulA at in vitro pull-down assay and these results indicated that pore I and loop2 are most responsiable for substrates binding. In addition, ClpY was capble of recognizing MBP-SulA wthout ATP to form SulA-ClpY complex and the ClpY hexamer can only bind a MBP-SulA molecule when ATP is present. These results also indicated that ClpY was capble to recognize SulA wthout ATP and formed SulA-ClpY complex for increasing its local surrounding substrate’s concentration and likely subsequently formed SulA-ClpY6 or SulA-ClpY12 complex docked with ClpQ for degradation when ATP is present. Y408A is not capble to form stable hexamer and ClpYQ oligomer maintains partial ATPase activity and degradation activity indicating that ClpYQ oligomerization is important for the enzyme activity.